In Situ Hybridization

KH Andy Choo, The Murdoch Institute, Melbourne, Australia Jeffrey M Craig, The Murdoch Institute, Melbourne, Australia Suzanne M Cutts, The Murdoch Institute, Melbourne, Australia Anthony WI Lo, The Murdoch Institute, Melbourne, Australia
In situ hybridization is based on the sequence-specific annealing of denatured nucleic acid strands. It combines the detection of specific nucleic acid sequences and their localization in relation to cellular or subcellular morphology.

Secondary article
Article Contents
. Introduction . Outline of Methods . Applications . Future Developments

Introduction
In the late 1960s, researchers began to realize that a great deal of important information could be gained by studying DNA sequences in their natural environment instead of looking at them purely as isolated molecules. A technique was needed that could take a piece of labelled DNA and hybridize it to its original location somewhere within a cell or tissue. Out of this need the technique of in situ hybridization (ISH) was born. ISH combines the detection of specic nucleic acid sequences and their localization in relationship to cellular or subcellular morphology. The technique is based on one of the fundamental physical properties of nucleic acids: sequence-specic annealing of denatured nucleic acid strands. A labelled fragment of nucleic acid acts as a probe and the whole procedure of hybridization is carried out on a slide where the target sequences are still in their native positions relative to the other cellular and molecular components that have been xed. Since their introduction, ISH and similar techniques have evolved from the use of radioactive detection to less hazardous nonradioactive means. The sensitivity of the technique has been improved so that single-copy genes can be detected. The standard lower limit of detection for the size of target nucleic acid sequence has been pushed downwards from megabases to a few kilobases. More recently, the dimension of detection has been further rened from a tissue or cellular level down to the molecular level. ISH has widespread applications for diagnosis and basic research.

malin. A permeabilization and unmasking procedure may be required to improve access of the probe to the target sequence, especially when the sample material has been treated with a stronger type of xative. This usually involves proteolytic digestions using enzymes such as proteinase K. Other methods may involve denaturants such as sodium bisulte and hydrochloric acid, or microwave and pressure cooking treatments.

Probe labelling
Molecules capable of forming stable duplexes with nucleic acids can be used as probes. These include DNA, RNA and, more recently, peptide nucleic acids (PNA). Nick translation, random-primed labelling, polymerase chain reaction (PCR), and terminal transferase reaction are well established methods of labelling. Alternatively, when oligonucleotides are used as probes, the labelled nucleotides can be incorporated directly during synthesis of the oligonucleotides. Nowadays, nonradioactive ligands, being more stable and less hazardous, are generally used. Isotopic labelling is mostly conned to RNA detection or when nonradioactive detection is not successful. The most commonly used ligands are digoxigenin- and biotinconjugated nucleotides. Fluorochromes such as uorescein or rhodamine conjugated directly onto nucleotides are increasingly used to further simplify the whole ISH process. ISH based on uorochrome detection is now widely referred to as FISH (uorescence in situ hybridization).

Outline of Methods
Sample slide preparations
Almost any form of tissue or cellular material can be used for ISH. These materials need to be xed onto a slide to preserve morphological detail (Figure 1). The xatives range from the milder reagents such as methanolacetic acid to stronger protein-crosslinking chemicals like for-

Denaturing and hybridization

The double helix and the secondary structures of the probe can easily be denatured by heating above melting temperature. The denaturation of the target sequences on the slides, on the other hand, requires more control since it is necessary to preserve the morphological details or other cellular components. Chemical and physical methods have been described, including strong alkaline treatment,
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In Situ Hybridization

annealing of the probe to the target sequences (Figure 2a(i)) while a low stringency allows the detection of targets of loosely similar sequences (Figure 2a(ii)).

Posthybridization washes
(a)

Washing removes excess probes that have not been bound to the target. Careful control of the stringency of hybridization and posthybridization washes ensures a good signal-to-noise ratio and improves the sensitivity and specicity of ISH.

Detection
The method of detection depends on the label used. Isotopic probes are detected by photographic emulsion coated onto the specimen. Probes labelled with uorochromes can be visualized under an epiuorescence microscope. Nonradioactive probes are detected indirectly by multiple layers of uorochrome-conjugated or enzymeconjugated antibodies. Images can be photographed directly or digitally captured by a cooled charged-coupled device (CCD) video camera. This camera system allows higher sensitivity and the convenience of immediate image analysis on a linked computer system.

(b)

State-of-the art techniques

Aside from the basic ISH procedure outlined, more complex techniques have recently evolved that are proving invaluable for research and diagnostics. These include bre FISH, comparative genome hybridization (CGH), in situ PCR, reverse painting, multicolour FISH, and chromosome bar-coding. In bre FISH, hybridization is performed on native DNA bres, thus increasing the resolution of ISH to the level of a few kilobases or less. CGH involves the use of two alternatively labelled sources of genomic DNA (e.g. tumour and normal), which are hybridized simultaneously onto normal metaphases; deviations from the combined colour ratio indicate areas of DNA amplications or deletions. In situ PCR is simply PCR performed on slide-bound cells or tissue sections sealed under a coverslip. PCR is also used to amplify small amounts of material from chromosomes that have been microdissected or ow-sorted; these probes can then be hybridized to normal metaphases. This technique has been used as a source of whole or regional chromosome paints to pinpoint the origins of marker chromosomes or rearranged chromosomal segments of unknown derivation in a process known as reverse painting. In multicolour FISH, a variety of probes, such as dierent chromosome paints, are labelled and detected in such a way that each appears as a dierent colour (Figures 2b(i) and 2b(ii)). Chromosome bar-coding takes a pool of chromosome band-specic or region-specic DNA probes and, via

(c)
Figure 1 The in situ hybridization procedure. (a) Fixation of cellular material (in this case metaphase chromosomes) onto a glass slide. (b) Denaturation of slide-bound nucleic acid and application of a labelled nucleic acid probe. (c) Hybridization of probe with complementary sequence and direct microscopic visualization of its cytogenetic location.

formamide denaturation at 70808C or dry heating at 948C. After the probe and the target sequences have been denatured, the two species are brought together in conditions favouring annealing. The stringency of hybridization can be controlled by the temperature, the concentration of formamide, and ionic strength of the reaction mixture. High temperature, high formamide concentration and low ionic strength favour specic annealing and vice versa. A high stringency ensures specic
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In Situ Hybridization

Figure 2 Examples of FISH applications where FISH images are obtained by digital capture. (a) Hybridization of normal human metaphase chromosomes (blue) using a centromeric a-satellite DNA probe (green) at (i) high stringency, showing specific centromeric signals on the cognate chromosomes 13 and 21, and (ii) low stringency, showing a generalized hybridization to all related centromeric a-satellite on every chromosome. (b) Multicolour (spectral) karyotyping, using differently coloured whole chromosome paints on (i) a normal human metaphase spread and (ii) a metaphase spread from a bladder cancer cell line showing multiple chromosome rearrangements (arrows). (Pictures courtesy of Hesed Padilla-Nash and Thomas Reid). (c) Trisomy detection. (i) Hybridization of a single-copy chromosome-21 YAC probe (yellow) to a Down syndrome metaphase chromosome spread showing specific mapping of the probe to band q22 (arrows) and the detection of three signals on interphase nuclei. (ii) Detection of three signals (green) on interphase nuclei using a chromosome 18-specific a-satellite probe at high stringency, corresponding to a diagnosis of trisomy 18.

multicolour FISH, provides colourful bar-codes with which to identify chromosomes.

pinpoint regions of the genome in which cancer-related genes reside and eventually lead to their discovery. There is also a wealth of evolutionary information to be gained from interspecies CGH studies.

Applications
Genome mapping
The Human Genome Project aims to map and sequence the entire human genome. ISH is being used to map unknown sequences to known landmarks at all levels: on metaphase chromosomes, interphase nuclei and DNA bres. Figure 2a shows an example of a chromosome landmark (repetitive centromeric a-satellite DNA) while Figure 2c(i) demonstrates the chromosomal localization of a unique sequence. Imbalances in the CGH pattern obtained from cancer cells, indicating DNA amplications or deletions, can also

Functional mapping
FISH can be used to map DNA and RNA to specic regions of metaphase chromosomes and interphase nuclei (which are usually three-dimensionally preserved). Families of repetitive sequences, genes, replicated DNA and amplied DNA have all been mapped en masse to chromosomes, as have sites of transgene or virus integration. Sequences can also be tracked through space and time; RNA can be followed within a nucleus, chromosome territories (and their changes throughout the cell cycle) can be mapped out within the cell or nucleus, and DNA
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In Situ Hybridization

sequences can be functionally linked with particular chromosomal substructures such as the centromere, telomere, chromosome scaold and sister-chromatid attachment sites. It is now possible to obtain from one slide data on chromosome constitution, gene expression (RNA), and protein expression at the tissue level or even at a single-cell level. An example of simultaneous DNA and protein detection is illustrated in Figure 3; the same underlying principle of dual-colour or multicolour labelling applies to DNARNA or RNAprotein detection.

Clinical applications
Clinical applications include preimplantation diagnosis (for in vitro fertilization), prenatal diagnosis, and the diagnosis and monitoring of neoplasia. A prime example is the use of a probe to the cancer gene HER2/neu, the amplication of which is linked with disease progression in breast cancer. ISH can be used to complement and even replace cytogenetic tests. Classical cytogenetic tests involve culturing cell suspensions and the collection and analysis of metaphase chromosomes. The limitations are numerous and include delay in obtaining results, inability to examine quiescent cells or those within a solid tumour, and the diculty of analysis of complex chromosome translocations. Using a combination of ISH techniques, all these limitations can be circumvented. In its quickest and simplest form, a probe can be hybridized to an interphase nucleus (Figure 2c(ii)), detected and recorded within just a few hours (compared with a few days with classical cytogenetics). In leukaemia, specic chromosome translocations (or the juxtaposition of genes at the translocation breakpoints) are associated with specic disease phenotypes and prognoses. An example is chronic myeloid leukaemia, in which the BCR gene on chromosome 22 is joined to the ABL gene on chromosome 9. Such translocations can be easily monitored in interphase nuclei.

The evolution of neoplastic clones, especially in solid tumours, can be monitored using CGH and multicolour chromosome painting. Figure 2b(ii) shows a metaphase spread from a bladder carcinoma cell line painted with whole human chromosome probes; a subset of abnormal chromosomes are easily identied because of their mixed colours. CGH and multicolour FISH can, in combination, identify most numerical and structural chromosome aberrations. The detection of specic RNA species is also useful for diagnosis of particular solid tumours, especially those of the endocrine system, in which tumour type can be linked with excess hormone production. The presence of viral DNA (by conventional ISH and in situ PCR) and RNA provides a sensitive assay for infection and the spread of viral diseases such as AIDS. ISH is also at the heart of new techniques for noninvasive prenatal diagnosis involving the detection of fetal cells in maternal blood. If successful, these techniques will replace procedures such as chorionic villus sampling (CVS) and amniocentesis that risk the health of the developing fetus.

Future Developments
New ISH probes are emerging at an ever-increasing rate. These need to be evaluated and their clinical applications carefully assessed. Subtelomeric probes are an example of such probes. These have been used to detect a whole new class of chromosome rearrangements involving loss or translocation of previously undetectable amounts of telomeric/subtelomeric DNA. The clinical applications of these probes are currently being evaluated. The resolution available for CGH is set to expand from the chromosome level down to that of a few kilobases; CGH has already been performed on individual DNA probes, either spotted in an array or stretched out, or combed onto microscope

Figure 3 Simultaneous DNA and protein detection. (a) A composite digital image for FISH using an a-satellite probe (green) specific for chromosomes 13 and 21 and the immunocytochemical fluorescence detection of a centromere-specific protein (pink) to demonstrate colocalization of the centromere protein and the DNA probe. (b) and (c) Using digital technology, the composite image can be split into its colour constituents to show signals for the chromosome 13- and 21-specific a-satellite sequences and the centromere protein, respectively.

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In Situ Hybridization

slides. The future will also see whole-mount FISH with RNA probes providing four-dimensional maps of gene expression (spatial distribution through time). The FISH toolbox contains an ever-expanding number of haptens for labelling and uorochromes for detection. Techniques are also evolving to speed up hybridization and detection. Improved tissue preparation and preservation, microscopy, digital imaging, data processing and storage complement these improvements. Commercially available labour-saving devices include probe labelling kits, readyfor-FISH probes and slides, slide-washing waterbaths, self-sealing coverslips, and chromosome-banding and antifade kits. Methods have also been developed to allow multiple sample deposition and hybridization on single slides. New techniques for signal amplication (e.g. using chemicals such as tyramide), primed in situ (PRINS) labelling, in situ PCR, PNA probing, and circularizing oligonucleotide probes are making possible detection of ever smaller targets, even single-nucleotide polymorphisms. The possibility of automation has been made a feasible goal by the development of synthetic membranes to replace glass slides. The technology developed for in situ hybridization has given rise to a whole new generation of techniques grouped under the description of DNA chip technology. DNA chips consist of microarrays of target DNA molecules on purpose-built glass slides or membranes. A complex set of probe molecules whose exact composition is unknown is hybridized to the target microarrays and hybridization is quantied with digital imaging. DNA chips containing up to a hundred thousand target molecules have already been synthesized, but the technology is still in its infancy. Some applications of this technology are in studies of gene expression and DNA sequencing. For the former, the

targets are cDNAs and the probe is total RNA. For the latter, the targets are dened oligonucleotides and the probe is the DNA to be sequenced. The oligonucleotide approach can also be used to detect all possible mutations in a given disease gene, which has obvious diagnostic benets. CGH at the level of a single gene is also a realistic goal using such techniques.

Further Reading
Bickmore WA (ed.) (1999) Chromosome Analysis: a Practical Approach. Oxford: Oxford University Press. Choo KHA (ed.) (1994) In Situ Hybridization Protocols. Totowa, NJ: Humana Press. du Sart D and Choo KHA (1996) The technique of in situ hybridization: principles and applications. In: Repley R and Walker JM (eds) Molecular Biomethods Handbook, pp. 697720. Totowa, NJ: Humana Press. Gole LA and Bongso A (1997) Fluorescence in situ hybridization some of its applications in clinical cytogenetics. Singapore Medical Journal 38: 497503. Heng HHQ, Spyropoulos B and Moens PB (1997) FISH technology in chromosome and genome research. BioEssays 19: 7584. Lichter P (1997) Multicolour FISHing: whats the catch? Trends in Genetics 13: 475479. McNicol AM and Farquharson MA (1997) In situ hybridization and its diagnostic applications in pathology. Journal of Pathology 182: 250 261. Raap AK (1998) Advances in in situ hybridization. Mutation Research 400: 287298. Ramsay G (1998) DNA chips: state-of-the art. Nature Biotechnology 16: 4044. Wienberg J and Stanyon R (1995) Chromosome painting in mammals as an approach to comparative genomics. Current Opinion in Genetics and Development 5: 792797. Wilkinson DG (ed.) (1999) In Situ Hybridization: A Practical Approach, 2nd edn. Oxford: Oxford University Press.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net